Project Summary World-wide sequencing efforts have identified recurrent novel genetic lesions associated with disease progression, inferred patterns of common co-occurrence, and characterized clonal evolution in response to treatments in chronic lymphocytic leukemia. Despite the wealth of available genetic information, these analyses all rely on statistical inference, thus limiting the possibility to link the genotype to the phenotype. Currently, understanding functions of high frequency genetic lesions and how they cooperate with their co-occurring mutations to cause CLL initiation and progression are not available. In this proposal, we aim to understand how two of the most recurrent genetic lesions in CLL (SF3B1 mutations and ATM deletions) mechanistically impact CLL initiation and progression with the overarching goal to understand CLL biology, generate novel murine model, and find new strategies for treating CLL with these lesions. This proposal is based on our finding that co-expression of mutated Sf3b1 with Atm deletion resulted in the development of clonal pathognomonic CD19+CD5+ B cells in blood, marrow and spleen at low penetrance in aged (18 months) mice, that can be propagated by in vivo passaging. Interestingly, whole-genome sequencing of DNA from murine CLL revealed recurrent chromosome amplifications, suggesting chromosome instability as a mechanism contributing to CLL in the mice with Sf3b1 mutation and Atm deletion. We now propose to investigate the hypothesis that SF3B1 mutations promote genomic instability through RNA splicing related R- loop formation while ATM deletion further augment genomic instability through decreasing R-loop associated DNA damage repair (Aim 1). We further hypothesize that modulation of the R-loop formation is contributing to the acceleration of CLL (Aim 2). Thus, targeting both RNA splicing and DNA damage response checkpoints are likely to provide synthetic cytotoxicity for CLLs with these lesions (Aim 3). Completion of the proposed work will create a fundamental foundation to explain how splicing factor mutations impact genomic instability and contribute to oncogenesis, and provide rationale for designing novel clinical trials in CLL patients with both SF3B1 mutations and ATM deletions.